Visual Tracking of Hydrogen Sulfide: Application of a Novel Lysosome-Targeted Fluorescent Probe for Bioimaging and Food Safety Assessment

The equilibrium state of hydrogen sulfide (H2S), a gaseous signaling molecule produced by lysosomal metabolites, in vivo is crucial for cellular function. Abnormal fluctuations in H2S concentration can interfere with the normal function of lysosomes, which has been closely linked to the pathogenesis of a variety of diseases. In view of this, a novel fluorescent probe Lyso-DPP based on 1,3,5-triarylpyrazolines was developed for the precise detection of H2S in lysosomes by using the hydrophilic morpholine moiety as a lysosomal targeting unit, and 2,4-dinitroanisole as a fluorescence-quenching and H2S-responsive unit. The probe cleverly combines the advantages of simple synthesis, sensitive blue fluorescence turn-on with a limit of detection, LOD, of 97.3 nM, good stability, and fast response time (10 min), which makes Lyso-DPP successful in portable monitoring of meat freshness in the form of test strips. Moreover, the excellent biocompatibility and precise targeting capability of the probe Lyso-DPP make it perform well in the monitoring of H2S in lysosomes, living cells, and zebrafish. This work not only provides new technical tools for food quality control but also paves up new ideas for early diagnosis and treatment of H2S-related diseases.


Introduction
Lysosomes are unique organelles in eukaryotic cells, known for their acidic environment (pH maintained between 4.5 and 5.5) and their abundance of hydrolytic enzymes.These enzymes not only serve as sites for intracellular waste disposal and degradation of biomolecules but also participate in a wide range of physiological processes, such as cellular autophagy, apoptosis, pathogen clearance, and cell signaling [1][2][3][4].The activity of hydrolytic enzymes within lysosomes is essential for the stability of the intracellular environment and the maintenance of cellular functions [5].Abnormalities in lysosomal function can interfere with critical physiological processes in organisms, leading to pathologies such as lysosomal storage diseases, renal diseases, inflammatory responses, and cancer [6,7].
During lysosomal metabolism, sulfur-containing amino acids, such as L-cysteine, generate hydrogen sulfide (H 2 S) through specific enzymatic reactions, for instance, by cystathionine-β-synthasecysteine (CBS), 3-mercaptopyruvate sulfotransferase (3-MST), and cystathionine-γ-lyase (CSE) [8,9].H 2 S, as an endogenous gaseous signaling molecule, participates in the regulation of cellular signaling and has a critical role in the homeostasis of a wide range of physiological and pathological processes.Maintaining homeostasis is crucial for human health [10][11][12].However, abnormal fluctuations in hydrogen sulfide concentration can cause lysosomal dysfunction and are also associated with pathological activity in Parkinson's disease, diabetes mellitus, Alzheimer's disease, gastric mucosal damage, and certain types of cancer, and these altered pathologies may have a profound Molecules 2024, 29, 3906 2 of 14 impact on an individual's longevity and quality of life [13,14].In addition, hydrogen sulfide, a toxic gas with a typical rotten egg odor, is released in elevated concentrations during food spoilage when bacteria decompose sulfides in food, a phenomenon that can serve as an important indicator of freshness in protein-rich meat products [15,16].In view of the important role of hydrogen sulfide in food quality and human health, it is particularly urgent to develop accurate assays to assess the levels of hydrogen sulfide in food and biological samples and even lysosomes.This will help improve food safety standards, enhance our awareness of the biological functions of hydrogen sulfide, and contribute to the scientific basis for the prevention and treatment of related diseases.
Fluorescence sensing technology has become an indispensable analytical tool in biomedical research due to its unique advantages.It not merely causes minimal damage to organisms but enables precise spatial localization and temporal tracking at the molecular level, demonstrating excellent spatial and temporal resolution [17][18][19][20].With the continuous progress of science and technology, fluorescence sensing technology has developed a variety of fluorophores, such as naphthalimide, coumarin, phenothiazine, BODIPY, etc.These fluorophores have different chemical properties and biocompatibility, which can meet the needs of different biological detection applications [21][22][23][24].Among these fluorophores, pyrazoline derivatives stand out for their excellent blue fluorescence emission, low cytotoxicity, high fluorescence quantum yield, and good photostability, which make them a highlight in the field of fluorescence sensing [25][26][27].Currently, many fluorescent probes for H 2 S detection in various complex environments and biological samples have been reported based on the aforementioned fluorophores utilizing reaction mechanisms like nitroxide reduction, azide reduction, nucleophilic substitution, and sulfurolysis (Table S1).Nevertheless, the available fluorescent probes are still insufficient in the face of the constant challenges of disease pathology research, especially for the specific detection of H 2 S in complex organelles such as lysosomes.As an important intracellular site of digestion and degradation, changes in H 2 S levels in lysosomes are closely associated with the development of a variety of diseases [28].Therefore, the development of fluorescent probes that can specifically target lysosomes and monitor the dynamic changes of H 2 S in lysosomes in real-time is of great significance for revealing the specific mechanisms of H 2 S in the pathological processes of diseases and exploring new therapeutic targets for diseases.
Based on the above, in order to overcome the limitations of the existing technology in the detection of H 2 S in lysosomes, the lysosome-targeted fluorescent probe Lyso-DPP was synthesized by using pyrazolines modified with a hydrophilic morpholine moiety as a fluorophore skeleton in combination with 2,4-dinitroanisole as a response moiety, and the probe itself exhibits a very low fluorescence background due to the fluorescencequenching effect of dinitroanisole.After triggering the nucleophilic substitution reaction of H 2 S, the rapid departure of the quencher 2,4-dinitroanisole resulted in a remarkable blue fluorescence response of the probe Lyso-DPP in aqueous solution, which was accompanied by a rapid turn-on of fluorescence (<10 min), a low limit of detection (97.3 nM), and superior specificity.Based on the favorable photophysical properties, we realized the detection of H 2 S in food spoilage by loading the probe into a portable paper-based assay.In bioimaging, Lyso-DPP successfully achieved outstanding visualization of exogenous H 2 S in HepG2 cells and endogenous H 2 S in HeLa cells and effectively monitored H 2 S at the zebrafish in vivo level.In addition, the fluorescence response of Lyso-DPP to H 2 S in a weakly acidic environment shows favorable adaptability, which enables the probe to adapt to the lysosomal environment and localize lysosomes more precisely, thus providing a powerful tool to precisely analyze the specific mechanism of H 2 S in cell metabolism, signaling, and even pathological processes.

Design and Synthesis of Lyso-DPP
In this study, we employed a multistep synthetic strategy to successfully construct a novel fluorescent probe Lyso-DPP (Scheme 1).First, we selected analytically pure 4-(4-morpholinyl)benzaldehyde and O-hydroxyacetophenone as starting materials and introduced a lysosome-targeted morpholino moiety into the molecular structure via an efficient condensation reaction.Further, Lyso-OH, a novel fluorophore based on the 1,3,5triarylpyrazoline backbone, was synthesized using the intramolecular ring-forming reaction of phenylhydrazine with compound 1. 2,4-dinitrophenyl ether was carefully selected as the quencher of the fluorophore and the functional group specifically recognizing H 2 S in the final stage of the synthesis of the probe Lyso-DPP.This design strategy ensures that the probe exhibits a low fluorescence state when it is not reacted with H 2 S, whereas once affected by H 2 S, the dissociation of 2,4-dinitrophenyl ether leads to the release of the fluorophore Lyso-OH, which enables the sensitive and selective detection of H 2 S. We performed an exhaustive structural characterization of the intermediate 1, the fluorophore Lyso-OH, and the final probe Lyso-DPP generated during the synthesis by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS) techniques (Figures S1-S9).

Design and Synthesis of Lyso-DPP
In this study, we employed a multistep synthetic strategy to successfully construct a novel fluorescent probe Lyso-DPP (Scheme 1).First, we selected analytically pure 4-(4morpholinyl)benzaldehyde and O-hydroxyacetophenone as starting materials and introduced a lysosome-targeted morpholino moiety into the molecular structure via an efficient condensation reaction.Further, Lyso-OH, a novel fluorophore based on the 1,3,5triarylpyrazoline backbone, was synthesized using the intramolecular ring-forming reaction of phenylhydrazine with compound 1. 2,4-dinitrophenyl ether was carefully selected as the quencher of the fluorophore and the functional group specifically recognizing H2S in the final stage of the synthesis of the probe Lyso-DPP.This design strategy ensures that the probe exhibits a low fluorescence state when it is not reacted with H2S, whereas once affected by H2S, the dissociation of 2,4-dinitrophenyl ether leads to the release of the fluorophore Lyso-OH, which enables the sensitive and selective detection of H2S.We performed an exhaustive structural characterization of the intermediate 1, the fluorophore Lyso-OH, and the final probe Lyso-DPP generated during the synthesis by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS) techniques (Figures S1-S9).

Photophysical Properties of Lyso-DPP
Following the successful synthesis of the fluorescent probe Lyso-DPP, we first probed the photophysical properties of Lyso-DPP using UV-Vis absorption and fluorescence emission spectra in EtOH-PBS buffer (v/v = 3/7, pH = 7.4, 20.0 mM) to evaluate its potential for fluorescence detection of H2S.As shown in Figure S10, we recorded the UV-Vis absorption spectra of Lyso-OH (10.0 µM) and Lyso-DPP (10.0 µM) in the presence or absence of H2S (100.0 µM).It was observed that the probe Lyso-DPP, which did not react with H2S, exhibited its maximum absorption peak at 372 nm.In contrast, when the probe Lyso-DPP reacted with H2S, its absorption peak underwent a slight blue shift to 359 nm, a change that coincided with the absorption peak of Lyso-OH.This blue shift of the absorption peak explains the mechanism of fluorescence enhancement of the probe Lyso-DPP upon reaction with H2S from a spectroscopic point of view.Namely, the dissociation of 2,4-dinitrophenyl leads to the exposure of the fluorophore Lyso-OH, which restores its fluorescence properties.
In further studies, we delved into the fluorescence response properties of the fluorescent probe Lyso-DPP to H2S.Through the analysis of fluorescence emission spectra (Figure 1A1,A2), the fluorescence signal of the probe Lyso-DPP was extremely weak when it did not react with H2S.However, with the gradual increase of H2S concentration, the fluorescence intensity of the probe at 475 nm exhibited an obvious concentration-dependent Scheme 1. Synthesis process of Lyso-DPP.

Photophysical Properties of Lyso-DPP
Following the successful synthesis of the fluorescent probe Lyso-DPP, we first probed the photophysical properties of Lyso-DPP using UV-Vis absorption and fluorescence emission spectra in EtOH-PBS buffer (v/v = 3/7, pH = 7.4, 20.0 mM) to evaluate its potential for fluorescence detection of H 2 S. As shown in Figure S10, we recorded the UV-Vis absorption spectra of Lyso-OH (10.0 µM) and Lyso-DPP (10.0 µM) in the presence or absence of H 2 S (100.0 µM).It was observed that the probe Lyso-DPP, which did not react with H 2 S, exhibited its maximum absorption peak at 372 nm.In contrast, when the probe Lyso-DPP reacted with H 2 S, its absorption peak underwent a slight blue shift to 359 nm, a change that coincided with the absorption peak of Lyso-OH.This blue shift of the absorption peak explains the mechanism of fluorescence enhancement of the probe Lyso-DPP upon reaction with H 2 S from a spectroscopic point of view.Namely, the dissociation of 2,4-dinitrophenyl leads to the exposure of the fluorophore Lyso-OH, which restores its fluorescence properties.
In further studies, we delved into the fluorescence response properties of the fluorescent probe Lyso-DPP to H 2 S. Through the analysis of fluorescence emission spectra (Figure 1A1,A2), the fluorescence signal of the probe Lyso-DPP was extremely weak when it did not react with H 2 S.However, with the gradual increase of H 2 S concentration, the fluorescence intensity of the probe at 475 nm exhibited an obvious concentration-dependent enhancement, which was increased 13-fold with the addition of 100.0 µM H 2 S.An excellent linear response relationship of Lyso-DPP to low concentrations of H 2 S in the range of 0.0-10.0µM was found by quantitative analysis (y = 24.518x+ 51.764, R 2 = 0.9934).The limit of detection (LOD) of Lyso-DPP for H 2 S was calculated to be 97.3 nM according to Equation 3δ/k (k represents the slope of the linear relationship exhibited between the Lyso-DPP fluorescence intensity and the change in hydrogen sulfide (H 2 S) concentration at a specific wavelength of 475 nm.δ is the standard deviation of the fluorescence intensity measurements of the blank sample.Calculated by repeating the measurement of the fluorescence spectrum of a blank sample of Lyso-DPP 10 times, we were able to assess the repeatability of the experiment and the level of background noise [29]).The above results indicate that the probe Lyso-DPP has high sensitivity and good potential for quantitative analysis in H 2 S detection.
enhancement, which was increased 13-fold with the addition of 100.0 µM H2S.A lent linear response relationship of Lyso-DPP to low concentrations of H2S in the r 0.0-10.0µM was found by quantitative analysis (y = 24.518x+ 51.764, R 2 = 0.993 limit of detection (LOD) of Lyso-DPP for H2S was calculated to be 97.3 nM accor Equation 3δ/k (k represents the slope of the linear relationship exhibited betw Lyso-DPP fluorescence intensity and the change in hydrogen sulfide (H2S) concen at a specific wavelength of 475 nm.δ is the standard deviation of the fluorescence in measurements of the blank sample.Calculated by repeating the measurement of orescence spectrum of a blank sample of Lyso-DPP 10 times, we were able to as repeatability of the experiment and the level of background noise [29]).The above indicate that the probe Lyso-DPP has high sensitivity and good potential for quan analysis in H2S detection.

Kinetic Study of Lyso-DPP
The fast response capability of small-molecule fluorescence sensors is crucial turing dynamic processes of bio-molecules during real-time monitoring and in sit ing.To fully evaluate the performance of the Lyso-DPP fluorescent sensor, we con an in-depth study of its temporal kinetic properties (Figure 1B).In the control exp without H2S, the fluorescence intensity of the blank probe solution did not change icantly during the observation period of 30 min, which indicates that Lyso-DPP ha lent chemical stability in aqueous solution.When different concentrations of H2 100.0 µM) were introduced, Lyso-DPP exhibited significant fluorescence enhan and the fluorescence intensity was gradually enhanced with the prolongation of

Kinetic Study of Lyso-DPP
The fast response capability of small-molecule fluorescence sensors is crucial for capturing dynamic processes of bio-molecules during real-time monitoring and in situ imaging.To fully evaluate the performance of the Lyso-DPP fluorescent sensor, we conducted an in-depth study of its temporal kinetic properties (Figure 1B).In the control experiment without H 2 S, the fluorescence intensity of the blank probe solution did not change significantly during the observation period of 30 min, which indicates that Lyso-DPP has excellent chemical stability in aqueous solution.When different concentrations of H 2 S (10.0-100.0µM) were introduced, Lyso-DPP exhibited significant fluorescence enhancement, and the fluorescence intensity was gradually enhanced with the prolongation of incubation time.In particular, Lyso-DPP achieved rapid fluorescence turn-on for all tested concentrations of H 2 S within 10 min and maintained its maximum fluorescence for the following 20 min.The rapid change in fluorescence of the test system reflects that Lyso-DPP can track dynamic changes in H 2 S concentration in real time.

pH Study of Lyso-DPP
In biological systems, different levels of microenvironments each maintain specific pH conditions.For this reason, we further investigated the ability of Lyso-DPP to detect H 2 S in a wide pH range of 2-12 (Figure 1C).The fluorescence signal of the blank probe set remained stable and quenched in aqueous solutions at pH 2-9.When the pH was 2-5, the detection ability of the probes for H 2 S was gradually enhanced until the fluorescence signal reached the maximum fluorescence intensity at pH = 5, and the fluorescence intensity remained stable in the pH range of 5-9.Furthermore, a significant increase in fluorescence intensity was observed when the pH was increased to 10-12, which might be due to the decomposition of the probe structure under alkaline conditions.The above results indicate that Lyso-DPP can achieve a precise response to H 2 S in a pH environment of 5-9.

Selectivity and Competition Study of Lyso-DPP
In order to deeply explore the specificity of the probe Lyso-DPP for the detection of H 2 S in complex biological systems, the response of Lyso-DPP to a variety of potential interferents was investigated, including cations (Na + , Mg 2+ , Ca 2+ , Zn 2+ , NH 4 + ), anions ), amino acids (Arg, Cys, Asn, Ile, Met, Ser, Val, Tyr, Glu, Phe, Lys, Hcy), and reactive oxygen species (ONOO − , H 2 O 2 , HCIO) [30][31][32].A significant enhancement of the fluorescence signal was not observed when the probe Lyso-DPP was exposed to a blank solution or contacted with the above-interfering substances.On the contrary, once Lyso-DPP was in contact with H 2 S, its fluorescence intensity at 475 nm increased significantly, a phenomenon visualized in Figure 1D.In addition, even in a complex environment where H 2 S coexists with other interferences, Lyso-DPP was still able to exhibit a pronounced blue fluorescence response.These results not only confirmed the high specificity of Lyso-DPP in detecting H 2 S but also demonstrated its potential application under complex physiological conditions.

Sensing Mechanism
Based on the spectral analysis and in-depth study of the existing literature [33,34], we hypothesized the response mechanism of the probe Lyso-DPP to H 2 S (Scheme 2) as follows: due to the Photoinduced Electron Transfer (PET) process, Lyso-DPP has almost no fluorescence signal when it does not react with H 2 S.However, when Lyso-DPP came into contact with H 2 S, the nucleophilic nature of H 2 S led to a rapid nucleophilic substitution reaction between it and Lyso-DPP, which led to the breakage of the dinitroanisole bond and was accompanied by the release of the fluorophore Lyso-OH.In order to verify this reaction mechanism, the high-performance liquid chromatography (HPLC) technique was employed to analyze the probe Lyso-DPP, the fluorophore Lyso-OH, and the components of the reaction mixture in detail (Figure 1F).The results showed that Lyso-DPP and Lyso-OH in the free state presented characteristic peaks on the chromatogram at 8.755 and 9.517 min, respectively.When the Lyso-DPP was mixed with H 2 S, we noticed that the characteristic peaks of the probe molecules originally located at 8.755 min gradually decreased with the increase of H 2 S concentration.In comparison, the peaks of the fluorophores located at 9.517 min increased accordingly.When an excess of H 2 S (10 equivalents) was added, only the characteristic peak at 9.517 min was observed in the reaction mixture.This further confirmed the completeness of the nucleophilic substitution reaction and the complete release of the fluorophore.Furthermore, to further verify the identity of the reaction product, high-resolution mass spectrometry (HRMS) analysis of the main peak detected at m/z = 400.2017was consistent with the expected mass of the fluorophore Lyso-OH (Figure S11), thus providing conclusive evidence for the reaction mechanism.
action product, high-resolution mass spectrometry (HRMS) analysis of the main pe tected at m/z = 400.2017was consistent with the expected mass of the fluorophore OH (Figure S11), thus providing conclusive evidence for the reaction mechanism.
Scheme 2. The proposed response pattern of Lyso-DPP to H2S.

Fluorescence Imaging in Live Cells
Given the excellent performance demonstrated by probe Lyso-DPP in aqueou tion, we were motivated to further explore its potential for imaging H2S in a cellula ronment.Prior to the study of its imaging capabilities, cytotoxicity was first evalua HepG2 hepatocytes and HeLa cervical cancer cells.MTT colorimetry was used to the potential effects of the probe on these cell lines (Figure S12).The experimental revealed that the survival rate of both cell types remained above 80% even at prob centrations as high as 40.0 µM, suggesting that Lyso-DPP has low cytotoxicity, whi vides an important prerequisite for the application of the probe in live cell imaging Subsequently, the ability of Lyso-DPP to detect exogenous H2S in HepG2 ce assessed by incubating HepG2 cells with different concentrations of H2S (10.0, 30 50.0 µM) for 30 min.The cells were imaged after being washed three times with ste PBS and introduced to Lyso-DPP (10.0 µM) to continue the incubation for 30 min. 2 showed that the fluorescence signal in the blue channel was enhanced with the in of H2S concentration, indicating that Lyso-DPP was able to effectively detect cellu ogenous H2S.

Fluorescence Imaging in Live Cells
Given the excellent performance demonstrated by probe Lyso-DPP in aqueous solution, we were motivated to further explore its potential for imaging H 2 S in a cellular environment.Prior to the study of its imaging capabilities, cytotoxicity was first evaluated on HepG2 hepatocytes and HeLa cervical cancer cells.MTT colorimetry was used to assess the potential effects of the probe on these cell lines (Figure S12).The experimental results revealed that the survival rate of both cell types remained above 80% even at probe concentrations as high as 40.0 µM, suggesting that Lyso-DPP has low cytotoxicity, which provides an important prerequisite for the application of the probe in live cell imaging.
Subsequently, the ability of Lyso-DPP to detect exogenous H 2 S in HepG2 cells was assessed by incubating HepG2 cells with different concentrations of H 2 S (10.0, 30.0, and 50.0 µM) for 30 min.The cells were imaged after being washed three times with sterilized PBS and introduced to Lyso-DPP (10.0 µM) to continue the incubation for 30 min.Figure 2 showed that the fluorescence signal in the blue channel was enhanced with the increase of H 2 S concentration, indicating that Lyso-DPP was able to effectively detect cellular exogenous H 2 S.
OH (Figure S11), thus providing conclusive evidence for the reaction mechanism.
Scheme 2. The proposed response pattern of Lyso-DPP to H2S.

Fluorescence Imaging in Live Cells
Given the excellent performance demonstrated by probe Lyso-DPP in aqueous tion, we were motivated to further explore its potential for imaging H2S in a cellular ronment.Prior to the study of its imaging capabilities, cytotoxicity was first evaluate HepG2 hepatocytes and HeLa cervical cancer cells.MTT colorimetry was used to a the potential effects of the probe on these cell lines (Figure S12).The experimental re revealed that the survival rate of both cell types remained above 80% even at probe centrations as high as 40.0 µM, suggesting that Lyso-DPP has low cytotoxicity, which vides an important prerequisite for the application of the probe in live cell imaging.
Subsequently, the ability of Lyso-DPP to detect exogenous H2S in HepG2 cells assessed by incubating HepG2 cells with different concentrations of H2S (10.0, 30.0 50.0 µM) for 30 min.The cells were imaged after being washed three times with steri PBS and introduced to Lyso-DPP (10.0 µM) to continue the incubation for 30 min.Fi 2 showed that the fluorescence signal in the blue channel was enhanced with the incr of H2S concentration, indicating that Lyso-DPP was able to effectively detect cellula ogenous H2S.Next, HeLa cells were selected to explore the ability of Lyso-DPP to detect cellular endogenous H 2 S (Figure 3).Since cysteine (Cys) can be converted to H 2 S intracellularly by the enzymatic reaction of cystathionine-γ-lyase (CSE), 200.0 µM Cys was chosen to incubate the cells for 1 h, and then Lyso-DPP (10.0 µM) was co-incubated with the cells for 30 min.A significantly enhanced fluorescence signal was observed when compared to the control group that was incubated with the probe Lyso-DPP only, indicating that Cys treatment significantly increased the level of endogenous H 2 S in the cells.To further demonstrate that the strong fluorescence signal generated by the Cys-incubated cells was due to the production of endogenous H 2 S, the same treatment described above was performed on the cells after selecting 1.0 mM propargylglycine (PAG, a common CSE inhibitor) for preincubation of the cells for 30 min, and a substantial attenuation of fluorescence intensity was observed.It was shown that Cys was able to increase the elevation of endogenous H 2 S levels as well as monitor the levels of endogenous and exogenous H 2 S by Lyso-DPP in the cells.
intensities in panel (A1-D3).n = 3, error bars were ±SD.Statistical analysis was perform one-way ANOVA.Among them, *** p < 0.001.Blue channel: λex = 405 nm, λem = 425-475 nm Next, HeLa cells were selected to explore the ability of Lyso-DPP to detect endogenous H2S (Figure 3).Since cysteine (Cys) can be converted to H2S intracellu the enzymatic reaction of cystathionine-γ-lyase (CSE), 200.0 µM Cys was chosen bate the cells for 1 h, and then Lyso-DPP (10.0 µM) was co-incubated with the ce min.A significantly enhanced fluorescence signal was observed when compare control group that was incubated with the probe Lyso-DPP only, indicating that C ment significantly increased the level of endogenous H2S in the cells.To further strate that the strong fluorescence signal generated by the Cys-incubated cells wa the production of endogenous H2S, the same treatment described above was pe on the cells after selecting 1.0 mM propargylglycine (PAG, a common CSE inhib preincubation of the cells for 30 min, and a substantial attenuation of fluorescenc sity was observed.It was shown that Cys was able to increase the elevation of end H2S levels as well as monitor the levels of endogenous and exogenous H2S by Ly in the cells.In order to deeply investigate the specific localization ability of Lyso-DPP fo lysosomes, a commercial lysosome-targeting dye (Lyso Tracker Red, 1.0 µM) was a control, and HeLa cells were co-incubated with Lyso-DPP (10.0 µM) for 30 min 4).A significant overlap in the spatial distribution of the red fluorescent signal fro Tracker Red and the blue fluorescent signal from Lyso-DPP was observed by fluo confocal microscopy imaging.This overlap suggests that the Lyso-DPP probe ca ically localize to lysosomes.The co-localization coefficient (Pearson's coefficient quantifies the extent of this co-localization, had a high value of 0.95, further con the specific localization of Lyso-DPP to lysosomes.In order to deeply investigate the specific localization ability of Lyso-DPP for H 2 S in lysosomes, a commercial lysosome-targeting dye (Lyso Tracker Red, 1.0 µM) was used as a control, and HeLa cells were co-incubated with Lyso-DPP (10.0 µM) for 30 min (Figure 4).A significant overlap in the spatial distribution of the red fluorescent signal from Lyso Tracker Red and the blue fluorescent signal from Lyso-DPP was observed by fluorescence confocal microscopy imaging.This overlap suggests that the Lyso-DPP probe can specifically localize to lysosomes.The co-localization coefficient (Pearson's coefficient), which quantifies the extent of this co-localization, had a high value of 0.95, further confirming the specific localization of Lyso-DPP to lysosomes.

Fluorescence Imaging in Zebrafish
In view of the high transparency of zebrafish embryos and the high homology between their genes and humans [35,36], 3-day-old zebrafish embryos were used as model organisms in this study to evaluate the bioimaging performance of Lyso-DPP and its sensitivity to the detection of H2S under in vivo conditions by fluorescence imaging.The experimental results exhibited (Figure 5) that the blue fluorescence intensity of zebrafish embryos was gradually enhanced with increasing H2S concentration (from 10.0 µM to 50.0 µM), which was similar to the previous observation in HepG2 cells.This increase in fluorescence intensity compared to the control group incubated with only 10.0 µM of Lyso-DPP probe indicates that Lyso-DPP can effectively detect exogenous H2S in zebrafish embryos.

Fluorescence Imaging in Zebrafish
In view of the high transparency of zebrafish embryos and the high homology between their genes and humans [35,36], 3-day-old zebrafish embryos were used as model organisms in this study to evaluate the bioimaging performance of Lyso-DPP and its sensitivity to the detection of H 2 S under in vivo conditions by fluorescence imaging.The experimental results exhibited (Figure 5) that the blue fluorescence intensity of zebrafish embryos was gradually enhanced with increasing H 2 S concentration (from 10.0 µM to 50.0 µM), which was similar to the previous observation in HepG2 cells.This increase in fluorescence intensity compared to the control group incubated with only 10.0 µM of Lyso-DPP probe indicates that Lyso-DPP can effectively detect exogenous H 2 S in zebrafish embryos.
Further, to explore the ability of Lyso-DPP to detect endogenous H 2 S, cysteine was employed as an inducer of endogenous H 2 S, and PAG was used to inhibit the conversion process of Cys to H 2 S. The experimental results indicated that 200.0 µM Cys treatment significantly increased the fluorescence intensity of zebrafish embryos, in contrast to the untreated control.Meanwhile, after PAG pretreatment, the fluorescence intensity of zebrafish embryos was significantly reduced even in the presence of Cys, suggesting that the enhancement of the fluorescence signal is indeed associated with the production of endogenous H 2 S.This finding further confirms the ability of Lyso-DPP to specifically detect endogenous and exogenous H 2 S in living zebrafish embryos.
embryos was gradually enhanced with increasing H2S concentration (from 10.0 µM to 50.0 µM), which was similar to the previous observation in HepG2 cells.This increase in fluorescence intensity compared to the control group incubated with only 10.0 µM of Lyso-DPP probe indicates that Lyso-DPP can effectively detect exogenous H2S in zebrafish embryos.

Detection of H 2 S and Food Samples by Means of Test Strips
The results of the spectral analysis confirmed that the probe Lyso-DPP is capable of quantitatively detecting H 2 S in the blue fluorescence channel.To validate the potential application of the probe in real-world environments, we further developed Lyso-DPP test strips for the detection of different concentrations of H 2 S (0.0-100.0 µM).The changes in fluorescence intensity of the test strips were recorded under irradiation by a 365 nm UV lamp (Figure S13).The experimental results showed that the blue fluorescence intensity of the test strips increased accordingly with the increase in H 2 S concentration, verifying that the Lyso-DPP test strips had a sensitive response to H 2 S.
Considering that the degradation of sulfides by microorganisms releases H 2 S during the deterioration process of meat, we further explored the application of Lyso-DPP test strips in meat freshness testing (Figure 6).For the experiment, we selected chicken, pork, beef, and fish samples of the same quality and placed them in Petri dishes containing Lyso-DPP test strips.At a constant temperature of 30 • C, we monitored the changes in the fluorescence intensity of the test strips.At the initial moment (0 h), the Lyso-DPP test strips did not display a fluorescent response, indicating that the food samples were in a fresh state.With time, a gradual increase in fluorescence intensity was observed, and this change was proportional to the amount of H 2 S released, thus reflecting a decrease in the freshness of the meat.This simple and rapid assay not only demonstrated the potential application of the Lyso-DPP probe in H 2 S detection but also provided a practical tool for food safety monitoring.
strips did not display a fluorescent response, indicating that the food samples were in a fresh state.With time, a gradual increase in fluorescence intensity was observed, and this change was proportional to the amount of H2S released, thus reflecting a decrease in the freshness of the meat.This simple and rapid assay not only demonstrated the potential application of the Lyso-DPP probe in H2S detection but also provided a practical tool for food safety monitoring.

Reagents and Apparatus
The solvents and reagents used in the experiments were purchased from commercial suppliers and were of analytically pure grade, which could be directly applied to the experimental process without additional purification steps.Meat and seafood samples were selected from the local market.Nuclear magnetic resonance (NMR) spectra of the compounds were acquired by Bruker Advance 600 MHz spectrometer.Ultraviolet-visible (UV-Vis) spectra and fluorescence spectral data were obtained by Unico UV-4802 spectrophotometer and Shimadzu RF-6000 fluorescence spectrophotometer.The bioimaging section was captured using a Zeiss (Oberkochen, Germany) LSM710 Wetzlar laser scanning confocal microscope.

Spectral Measurement
Lyso-DPP was prepared by dissolving it in DMSO to form a probe stock solution at a concentration of 0.3 mM, which was further dissolved in a mixed ethanol/PBS solution (v/v = 3/7, pH = 7.4) to obtain the final probe solution.1.0 mM Na 2 S was solubilized in ultrapure water to form a stabilized donor solution of H 2 S.This donor solution was then diluted to a series of different concentration gradients, depending on the experimental requirements, for fluorescence spectroscopy at an excitation wavelength of 360 nm, with an excitation slit width of 5 nm and an emission slit width of 10 nm.Other interferences (Na + , Mg 2+ , Ca 2+ , Zn 2+ , NH 4 + , Arg, Cys, Asn, Ile, Met, Ser, Val, Tyr, Glu, Phe, Lys, Hcy, NO 3 − , ONOO − , SO 3 2− , CO 3 2− , ClO 4 − , H 2 O 2 , HCIO) were prepared as stock solutions in pure water at a concentration of 3.0 mM for selection and competition experiments.

Cell Culture and Incubation
HepG2 cells and HeLa cells were cultured in DEME medium supplemented with 10% fetal bovine serum at 37 • C with 5% CO 2 .Cells were inoculated into 96-well plates and incubated for 24 h.Cells were adhered to the wall and then incubated with different concentrations of probe solution (0.0, 10.0, 20.0, 30.0, 40.0 µM) for 24 h.Cells were washed three times with sterilized PBS and then added with MTT solution (5 mL/mg) and incubated for 4 h.Cell activity was analyzed quantitatively by measuring the absorbance at 490 nm.
In this study, HepG2 cells were selected to assess the ability to detect exogenous H 2 S. In control experiments, cells were incubated with a 10.0 µM fluorescent probe for 30 min only.Comparatively, in the experimental group, cells were first pre-treated with 10.0-50.0µM H 2 S solution for 30 min to simulate the exposure environment of exogenous H 2 S, followed by incubation with the same concentration of probe for 30 min to observe the fluorescence response of the probe to H 2 S.
For the detection of endogenous H 2 S, HeLa cells were selected for imaging experiments.The incubation conditions of the control group were identical to those of the control group for exogenous H 2 S. In the experimental group, we used two different treatments.First, cells were pretreated with 200.0 µM Cys for 1 h to promote endogenous H 2 S production and then incubated with a fluorescent probe for imaging.Second, another set of HeLa cells was preloaded with propargylglycine (PAG, 1.0 mM, an inhibitor of CSE enzyme) for 30 min, after which they continued to receive treatments of Cys (200.0 µM) and Lyso-DPP (10.0 µM) for 1 h and 30 min, respectively.Prior to fluorescence imaging, all cell samples were washed three times with sterile phosphate buffer solution.

Zebrafish Incubation
Three-day-old zebrafish provided by Nanjing EzeRinka Biotechnology Co., Ltd (Nanjing, China) and used for in vivo imaging of H 2 S. Zebrafish were first incubated with only 10.0 µM probe solution for 30 min as a control.For the determination of exogenous H 2 S, zebrafish were stained with 10.0 µM probe solution for 30 min based on incubation with different concentrations of H 2 S (30.0 µM, 50.0 µM), whereas for endogenous H 2 S, zebrafish were divided into two groups, one group of zebrafish was treated with 200.0 µM Cys for 1 h, and the other group was treated with 200.0 µM Cys in zebrafish loaded with 1.0 mM PAG for 30 min, followed by incubation of both groups with 10.0 µM probe solution.The incubation was followed by three washes with sterile PBS before imaging.All animal-related experimental manipulations strictly followed the rigorous review and formal approval by the Animal Ethics Committee of Qiqihar Medical University (approval number QMU-AECC-2024-111).

Test Strips Test Processing
To explore the application of the Lyso-DPP probe in the detection of H 2 S, filter papers were first cut into rectangular shapes of 1 cm width and 3 cm length.Next, these paper strips were immersed in Lyso-DPP solution at a concentration of 50.0 µM for 20 min to fully adsorb the probe molecules.Subsequently, the paper strips were removed and placed in air to dry to ensure that the probe molecules were uniformly immobilized on the filter paper.Following this, two different sets of experimental treatments were performed.First, the dried filter paper was incubated with different concentrations of H 2 S (0.0-100.0 µM) for 20 min to simulate the fluorescence response at different H 2 S concentrations in order to construct a fluorescence colorimetric card of H 2 S for quantitatively analyzing the concentration of H 2 S in the actual environment.Next, the same filter paper was directly exposed to fresh meat and seafood samples to verify the probe's ability to detect food freshness.The fluorescence changes of the filter paper were observed under a 365 nm handheld UV lamp.

Conclusions
In summary, an H 2 S "turn-on" fluorescent probe Lyso-DPP based on pyrazoline derivatives was designed, and the probe itself appears fluorescence-quenched due to the PET effect.When contacted with H 2 S, the probe undergoes a rapid nucleophilic substitution reaction, leading to the appearance of strong blue fluorescence at 475 nm.This fluorescence enhancement not only provides an intuitive and sensitive signal for the detection of H 2 S but also greatly improves the convenience and sensitivity of H 2 S monitoring during meat spoilage through the application of test paper format.In addition, thanks to the low cytotoxicity and high specificity for H 2 S, Lyso-DPP is able to accurately reflect the concentration changes of H 2 S in living cells as well as zebrafish, demonstrating excellent imaging results.Notably, the clever incorporation of the morpholine moiety in Lyso-DPP endowed it with precise lysosomal targeting ability, which enabled the probe to show extraordinary advantages in the detection of H 2 S in lysosomes.The present study not only provides a new tool for real-time monitoring of H 2 S but also offers a new perspective for a deeper understanding of the role of H 2 S in cellular physiology and pathological processes through precise lysosomal targeting.

Supplementary Materials:
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules29163906/s1,Table S1.Comparison of the proposed probe with other reported fluorescence probes for the detection of H 2 S. Figure S1. 1 H NMR spectrum of compound 1 in DMSO-d6. Figure S2. 13C NMR spectrum of compound 1 in DMSO-d6. Figure S3.HRMS spectrum of compound 1.

Figure 4 .
Figure 4. Co-localization imaging of Lyso-DPP and Lyso Tracker Red in HeLa cells.(A) Fluorescence imaging of Lyso Tracker Red at 580-620 nm; (B) Fluorescence imaging of Lyso-DPP at 425-475 nm.(C) Merged fluorescence image.(D) Bright image.(E) Correlation analysis of fluorescence intensity between (A) and (B).(F) Fluorescence intensity distribution of the red channel (Lyso Tracker Red) and the blue channel (Lyso-DPP) on the yellow line.

Figure 4 .
Figure 4. Co-localization imaging of Lyso-DPP and Lyso Tracker Red in HeLa cells.(A) Fluorescence imaging of Lyso Tracker Red at 580-620 nm; (B) Fluorescence imaging of Lyso-DPP at 425-475 nm.(C) Merged fluorescence image.(D) Bright image.(E) Correlation analysis of fluorescence intensity between (A) and (B).(F) Fluorescence intensity distribution of the red channel (Lyso Tracker Red) and the blue channel (Lyso-DPP) on the yellow line.